Biochemical characterization of the interaction between HspA1A and phospholipids.

Seventy-kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis. Apart from their indispensable roles in protein homeostasis, specific Hsp70s localize at the plasma membrane and bind to specific lipids. The interaction of Hsp70s with lipids has direct physiological outcomes including lysosomal rescue, microautophagy, and promotion of cell apoptosis. Despite these essential functions, the Hsp70-lipid interactions remain largely uncharacterized. In this study, we characterized the interaction of HspA1A, an inducible Hsp70, with five phospholipids. We first used high concentrations of potassium and established that HspA1A embeds in membranes when bound to all anionic lipids tested. Furthermore, we found that protein insertion is enhanced by increasing the saturation level of the lipids. Next, we determined that the nucleotide-binding domain (NBD) of the protein binds to lipids quantitatively more than the substrate-binding domain (SBD). However, for all lipids tested, the full-length protein is necessary for embedding. We also used calcium and reaction buffers equilibrated at different pH values and determined that electrostatic interactions alone may not fully explain the association of HspA1A with lipids. We then determined that lipid binding is inhibited by nucleotide-binding, but it is unaffected by protein-substrate binding. These results suggest that the HspA1A lipid-association is specific, depends on the physicochemical properties of the lipid, and is mediated by multiple molecular forces. These mechanistic details of the Hsp70-lipid interactions establish a framework of possible physiological functions as they relate to chaperone regulation and localization.